Substituted N-acetyl-L-cysteine derivatives and related compounds

ABSTRACT

Novel substituted N-acetyl-L-cysteine (NAC) derivatives and related compounds and methods of using these compounds for the treatment of diseases and/or conditions, including but not limited to diseases and/or conditions of, or involving, the Central Nervous System (CNS), including schizophrenia adrenoleukodystrophy, mitochondrial diseases (e.g. Leigh syndrome, Alpers′ disease, and MELAS), Huntington&#39;s disease, trichotillomania, HIV-associated neurocognitive disorder, hypoxic-ischemic encephalopathy, drug craving, and drug addiction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. patent applicationSer. No. 14/535,855, filed Nov. 7, 2014, which claims the benefit ofU.S. Provisional Application 61/902,669, filed Nov. 11, 2013, and U.S.Provisional Application 61/902,052, filed Nov. 8, 2013, the disclosureof each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to novel substituted N-acetyl-L-cysteine (“NAC”)derivatives and related compounds and methods of using these compoundsfor the treatment of diseases and/or conditions, including but notlimited to diseases and/or conditions of, or involving, the CentralNervous System (CNS), including schizophrenia adrenoleukodystrophy,mitochondrial diseases (e.g. Leigh syndrome, Alpers′ disease, andMELAS), Huntington's disease, trichotillomania, HIV-associatedneurocognitive disorder, hypoxic-ischemic encephalopathy, drug craving,and drug addiction.

BACKGROUND OF THE INVENTION

Schizophrenia is a debilitating disorder afflicting 1% of the world'spopulation. The development of effective medications to treatschizophrenia relies on advances in characterizing the underlyingpathophysiology. Chlorpromazine and other phenothiazines are consideredfirst generation antipsychotics (termed “typical antipsychotics”) usefulin the treatment of schizophrenia.

Adrenoleukodystrophy, or X-linked adrenoleukodystrophy, is an inheritedlife-threatening metabolic rare disease. It primarily affectsmyelination throughout the nervous system, the adrenal cortex, and theLeydig cells in the testes where very-long chain fatty acids accumulate.The adrenoleukodystrophy patient population is heterogeneous, withclinical phenotypes that include progressive neurodegenerative declineleading to vegetative state in children (childhood cerebral X-linkedadrenoleukodystrophy). Treatments for adrenoleukodystrophy are includehematopoietic stem cell transplant, which results in a high-survivalrate (92% 5-year survival; Peters et al., “Cerebral X-linkedadrenoleukodystrophy: the international hematopoietic celltransplantation experience from 1982 to 1999,” Blood, 104: 881-888(2004)); however, this treatment is limited to and effective with only asmall adrenoleukodystrophy subpopulation, with success typically comingwhen it is performed in the early stages of the disease.

Adrenoleukodystrophy patients have one or more mutations to the ABCD1gene, which encodes the peroxisomal ATP-binding cassette transporter.Subsequently, very-long chain fatty acids build up in affected cells,leading to oxidative stress and eventually metabolic failure resultingin cell death, features common to human patients and animal models(Fourcade, S, et al. “Early oxidative damage in neurodegenerationUnderlying X-adrenoleukodystrophy,” Human Molecular Genetics, 17:1762-1773 (2008); López-Erauskin, J, et al., “Antioxidants halt axonaldegeneration in a mouse model of X-adrenoleukodystrophy,” Annals ofNeurology, 70: 84-92 (2011)).

Antioxidant therapy is a promising therapy for the treatment ofadrenoleukodystrophy and other diseases that involve oxidative stress.At the cellular level, antioxidants have been demonstrated to normalizebiomarkers of oxidative stress. N-acetylcysteine (“NAC”) is a prodrug ofcysteine, which serves as the limiting reagent in the synthesis ofglutathione, the body's major antioxidant. When given as an adjuvanttherapy to hematopoietic stem cell transplant in advanced stagechildhood cerebral X-linked adrenoleukodystrophy patients, patientsurvival outcome greatly improves with NAC treatment (Miller, W, et al.,“Outcomes after allogeneic hematopoietic cell transplantation forchildhood cerebral adrenoleukodystrophy: the largest single-institutioncohort report,” Blood, 118: 1971-1978 (2011); Tolar, J, et al.,“N-acetyl-L-cysteine improves outcome of advanced cerebraladrenoleukodystrophy.” Bone Marrow Transplant, 39: 211-215 (2007)).However, brain penetrance is low and the long-term risks and benefitsremain unknown.

Inherited mitochondrial diseases (e.g. Leigh syndrome, Alpers′ disease,and MELAS) affecting the CNS are highly variable, and often result inthe progressive loss, or dysfunction, of neurons or neuroglial cells. Inmany cases, the pathogenesis is a result of disruption of mitochondrialrespiratory chain processes, which can then increase the generation ofreactive oxidative species (ROS), due to mutations in mitochondrial ornuclear DNA. Antioxidant therapy, specifically N-acetylcysteine, acts todecrease ROS and increases glutathione levels, which concomitantlyincrease cell survival and function.

A range of other diseases share common pathophysiology with abnormalglutamate signaling and heightened levels of oxidative stress,particularly with System x_(c)-, a glutamate-cystine antiporter.Therefore, by engaging a single target, e.g. System x_(c)-, which is atthe junction of two distinct metabolic pathways, NAC, NAC derivativesand related molecules, may effectively treat these wide-ranging, andseemingly unrelated, diseases and disorders. This has been partiallydemonstrated in clinical study with NAC treatment of trichotillomania(Grant, J E, et al., “N-Acetylcysteine, a Glutamate Modulator, in theTreatment of Trichotillomania,” Arch Gen Psychiatry, 66: 756-763(2009)). System x_(c)-, NAC, and disturbances in glutamate signaling andoxidative stress are also linked to other diseases that include, but arenot limited to, Huntington's disease (Frederick, N M, et al.,“Dysregulation of system xc(-) expression induced by mutant huntingtinin a striatal neuronal cell line and in R6/2 mice,” Neurochem. Int.,2014; 76: 59-69), hypoxic-ischemic encephalopathy (Wang, X, et al.,“N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemicbrain injury,” Ann. Neurol., 61: 263-271 (2007)), HIV-associatedneurocognitive disorder (Vázquez-Santiago, F J, et al., “Glutamatemetabolism and HIV-associated neurocognitive disorders,” J. Neurovirol.,20: 315-331 (2014)).

Schizophrenia may be associated with abnormal glutamate signaling anddiminished glutathione levels. Impaired cystine-glutamate antiporteractivity can lead to increased oxidative stress and depletedglutathione, as well as abnormal glutamate neurotransmission, synapticconnection, and gene expression, all of which are observed inschizophrenia. In addition, impaired cystine-glutamate antiporteractivity and faulty glutamate neurotransmission bear on the issue ofuncontrolled drug use, i.e., drug addiction.

Cysteine prodrugs, such as NAC, drive cystine-glutamate exchange byapparently elevating extracellular cystine levels, thereby creating asteep cystine concentration gradient.

However, alternatives to NAC are needed. NAC undergoes extensive firstpass metabolism requiring the usage of high doses that limit the utilityof the drug and, potentially, increase the chances of side effects dueto the buildup of metabolized by-products. The compounds of the presentinvention are designed to substantially avoid the problem of first passmetabolism and therefore exhibit increased efficacy as compared to NACand other prior cysteine prodrugs. In addition, NAC demonstrates poorCNS penetration due to an inability to cross the blood brain barrier.

Accordingly, there is a need for novel compounds that would have areduced incidence of problems associated with NAC. The compounds of thepresent invention are designed to substantially avoid the problems offirst pass metabolism and poor CNS bioavailability, thereby exhibitingincreased efficacy as compared to NAC and other prior cysteine prodrugs.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a compound offormula I:

where R₂ is selected from the group consisting of: NH₂, (C₁₋₈ alkyl)O—,and OH.

In one embodiment, any one of C₂ through C₇ in the alkyl chain can besubstituted with either nitrogen or oxygen.

In a preferred embodiment R₂ is t-butyl-O— or (CH₃)(CH₂)O—,

R₃ is selected from the group consisting of:

R₄ is selected from the group consisting of: H and

In particular, the invention provides the following compounds:

In another aspect, the present invention is directed to a compound offormula II:

The invention also encompasses pharmaceutically acceptable salts, estersor prodrugs of the provided compounds.

In another aspect, the invention is directed to a method of treating adisease or condition in a subject comprising administering to thesubject a therapeutically effective amount of a compound of any ofFormulas I-II or a pharmaceutically acceptable salt, ester or prodrugthereof. The preferred route of administering to the subject is via oraldelivery. Preferably, diseases or conditions treatable with thecompounds of the present invention are related to the CNS.

In a preferred embodiment, the disease is schizophrenia.

In another aspect, the invention provides a method of treating drugcraving in a subject comprising administering to the subject atherapeutically effective amount of a compound of any of Formulas I-IIor a pharmaceutically acceptable salt, ester or prodrug thereof. Thepreferred route of administering to the subject is via oral delivery.

The invention further encompasses pharmaceutical compositions containinga compound of any of Formulas I-II or a pharmaceutically acceptablesalt, ester or prodrug thereof in combination with apharmaceutically-acceptable carrier.

Methods of formulating/manufacturing such pharmaceutical compositions(alternatively termed “medicaments”) for the treatment of a disease orcondition in a subject are also within the invention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are graphical representations of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-2022 as a percentage of the control.

FIGS. 2A-2D are graphical representations of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-2023 as a percentage of the control.

FIG. 3 is a graphical representation of the ¹⁴C-cystine uptake byPro-2024 as a percentage of the control.

FIG. 4 is a graphical representation of the ¹⁴C-cystine uptake byPro-3010 as a percentage of the control.

FIGS. 5A-5D are graphical representations of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-4006 as a percentage of the control.

FIGS. 6A-6D are graphical representation of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-4011 as a percentage of the control.

FIGS. 7A-7D are graphical representations of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-4047 as a percentage of the control.

FIGS. 8A-8D are graphical representation of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-4051 as a percentage of the control.

FIGS. 9A-9D are graphical representation of the ¹⁴C-cystine uptake and³H-glutamate release by Pro-4051a as a percentage of the control.

FIG. 10 is a graphical representation of the amount of intracellularcysteine for Pro-2023.

FIG. 11 is a graphical representation of the amount of intracellularcysteine for Pro-4006.

FIG. 12 is a graphical representation of the amount of intracellularcysteine for Pro-4011.

FIG. 13 is a graphical representation of the amount of intracellularcysteine for Pro-4047.

FIG. 14 is a graphical representation of the amount of intracellularcysteine for Pro-4051.

FIG. 15 is a graphical representation of the amount of intracellularcysteine for Pro-4051a.

FIG. 16 is a graphical representation of prepulse inhibition forPro-2023.

FIG. 17 is a graphical representation of prepulse inhibition forPro-4047.

FIG. 18 is a graphical representation of prepulse inhibition forPro-4051.

FIG. 19 is a graphical representation the total time Pro-2023 treatedrats spent on the open arms of the elevated-plus maze.

FIG. 20 is a graphical representation the total time Pro-4047 treatedrats spent on the open arms of the elevated-plus maze.

FIG. 21 is a graphical representation the total time Pro-4051 treatedrats spent on the open arms of the elevated-plus maze.

FIG. 22 is a graphical representation the total time Pro-4051a treatedrats spent on the open arms of the elevated-plus maze.

FIG. 23 is a graphical representation of levels of NAC present in thebrain after oral administration of Pro-2023.

FIG. 24 is a graphical representation of levels of NAC present in thebrain after oral administration of Pro-2024.

FIG. 25 is a graphical representation of levels of NAC present in thebrain after oral administration of Pro-4051.

FIG. 26 is a graphical representation of levels of glutathione presentin the brain after oral administration of Pro-2023.

FIG. 27 is a graphical representation of levels of glutathione presentin the brain after oral administration of Pro-2024.

FIG. 28 is a graphical representation of levels of glutathione presentin the brain after oral administration of Pro-3010.

FIG. 29 is a graphical representation of levels of glutathione presentin the brain after oral administration of Pro-4051.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used, unless otherwise described.

The term “prodrug” refers to compounds, including monomers and dimers ofthe compounds of the invention, which have cleavable groups and becomeunder physiological conditions compounds which are pharmaceuticallyactive in vivo.

The term “ester” refers to compounds having a generic structure ofRCO₂R′, where R and R′ are the organic parts of the carboxylic acid andalcohol respectively.

The term “subject” includes mammals. Mammals include but are not limitedto humans. The terms “patient” and “subject” are used interchangeably.

The term “therapeutically effective amount” means the amount of acompound that, when administered to a subject for treating a disease ordisorder, is sufficient to effect such treatment for the disease ordisorder. The “therapeutically effective amount” can vary depending onthe compound, the disease or disorder and its severity, and the age,weight, etc., of the subject to be treated.

The terms “treating” or “treatment” of any disease or disorder refer, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingor reducing the development of the disease or at least one of theclinical symptoms thereof). In another embodiment “treating” or“treatment” refers to ameliorating at least one physical parameter,which may not be discernible by the subject. In yet another embodiment,“treating” or “treatment” refers to modulating the disease or disorder,either physically, (e.g., stabilization of a discernible symptom),physiologically, (e.g., stabilization of a physical parameter), or both.In yet another embodiment, “treating” or “treatment” refers to delayingthe onset of the disease or disorder, or even preventing the same.

The term “alkyl” is intended to mean a straight chain or branchedaliphatic group having from 1 to 12 carbon atoms, alternatively 1-8carbon atoms, and alternatively 1-6 carbon atoms. In some embodiments,the alkyl groups have from 2 to 12 carbon atoms, alternatively 2-8carbon atoms and alternatively 2-6 carbon atoms. Examples of alkylgroups include, without limitation, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

In general, unless indicated otherwise, a chemical group referred toanywhere in the specification can be optionally substituted.

In another aspect, the present invention is directed to compounds offormula I:

where R₂ is selected from the group consisting of: NH₂, (C₁₋₈ alkyl)O—,and OH.

In one embodiment, any one of C₂ through C₇ in the alkyl chain can besubstituted with either nitrogen or oxygen.

In a preferred embodiment R₂ is t-butyl-O— or (CH₃)(CH₂)O—,

R₃ is selected from the group consisting of:

R₄ is selected from the group consisting of: H and

In particular, the invention provides the following compounds:

In another aspect, the present invention is directed to compounds offormula II:

The invention also encompasses pharmaceutically acceptable salts, estersor prodrugs of the provided compounds.

Certain compounds of the invention may exist in different isomeric (e.g.enantiomers and distereoisomers) forms. The invention contemplates allsuch isomers both in pure form and in admixture, including racemicmixtures. Enol forms are also included.

The compounds of the invention can exist in unsolvated as well assolvated forms, including hydrated forms, e.g., hemi-hydrate. Ingeneral, the solvated forms, with pharmaceutically acceptable solventssuch as water, ethanol, and the like are equivalent to the unsolvatedforms for the purposes of the invention.

Certain compounds of the invention also form pharmaceutically acceptablesalts, e.g., acid addition salts. The phrase “pharmaceuticallyacceptable salt” means those salts which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand lower animals without undue toxicity, irritation, allergic responseand the like and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well-known in the art. Forexample, S. M. Berge et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 66: 1 et seq. (1977). Forexample, for acid addition salts the nitrogen atoms may form salts withacids. Examples of suitable acids for salt formation are hydrochloric,sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,furmaric, succinic, ascorbic, maleic, methanesulfonic and other mineralcarboxylic acids well known to those in the art. The salts are preparedby contacting the free base form with a sufficient amount of the desiredacid to produce a salt in the conventional manner. The free base formsmay be regenerated by treating the salt with a suitable dilute aqueousbase solution such as dilute aqueous hydroxide potassium carbonate,ammonia, and sodium bicarbonate. The free base forms differ from theirrespective salt forms somewhat in certain physical properties, such assolubility in polar solvents, but the acid salts are equivalent to theirrespective free base forms for purposes of the invention. (See, forexample S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66:1-19 (1977) which is incorporated herein by reference.)

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, from acombination of the specified ingredients in the specified amounts.

The compounds of the present invention can be used in the form ofpharmaceutically acceptable salts derived from inorganic or organicacids. The salts can be prepared in situ during the final isolation andpurification of the compounds of the invention or separately by reactinga free base function with a suitable organic acid. Representative acidaddition salts include, but are not limited to acetate, adipate,alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate,hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate),lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate,oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate,glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, thebasic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which canbe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolationand purification of compounds of this invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylammonium,dimethylammonium, trimethylammonium, triethylammonium, diethylammonium,and ethylammonium among others. Other representative organic aminesuseful for the formation of base addition salts include ethylenediamine,ethanolamine, diethanolamine, piperidine, piperazine and the like.

Dosage forms for topical administration of a compound of this inventioninclude powders, sprays, ointments and inhalants. The active compound ismixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives, buffers or propellants which canbe required. Ophthalmic formulations, eye ointments, powders andsolutions are also contemplated as being within the scope of thisinvention.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention can be varied so as to obtain an amountof the active compound(s) which is effective to achieve the desiredtherapeutic response for a particular patient, compositions and mode ofadministration. The selected dosage level will depend upon the activityof the particular compound, the route of administration, the severity ofthe condition being treated and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the present invention can be employedin pure form or, where such forms exist, in pharmaceutically acceptablesalt, ester or prodrug form. Alternatively, the compound can beadministered as a pharmaceutical composition containing the compound ofinterest in combination with one or more pharmaceutically acceptableexcipients.

The phrase “therapeutically effective amount” of the compound of theinvention means a sufficient amount of the compound to treat disorders,at a reasonable benefit/risk ratio applicable to any medical treatment.It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds of this invention administered toa human or lower animal may range from about 0.0001 to about 2000mg/kg/day. For purposes of oral administration, more preferable dosescan be in the range of from about 0.001 to about 15 mg/kg/day, with themost preferable dose being in the range of from about 0.001 to about 5mg/kg/day. If desired, the effective daily dose can be divided intomultiple doses for purposes of administration; consequently, single dosecompositions may contain such amounts or submultiples thereof to make upthe daily dose.

The present invention also provides pharmaceutical compositions thatcomprise compounds of the present invention formulated together with oneor more pharmaceutically acceptable carriers. The pharmaceuticalcompositions can be specially formulated for oral administration insolid or liquid form, for parenteral administration or for rectaladministration.

The pharmaceutical compositions of this invention can be administered tohumans and other mammals orally, rectally, parenterally,intracisternally, intravaginally, transdermally (e.g. using a patch),transmucosally, sublingually, pulmonary, intraperitoneally, topically(as by powders, ointments or drops), bucally or as an oral or nasalspray. The terms “parental” or “parenterally,” as used herein, refers tomodes of administration which include intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous and intraarticular injectionand infusion.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a component of the present invention and aphysiologically tolerable diluent. The present invention includes one ormore compounds as described above formulated into compositions togetherwith one or more physiologically tolerable or acceptable diluents,carriers, adjuvants or vehicles that are collectively referred to hereinas diluents, for parenteral injection, for intranasal delivery, for oraladministration in solid or liquid form, for rectal or topicaladministration, among others.

Compositions suitable for parenteral injection may comprisephysiologically acceptable, sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), vegetable oils (such asolive oil), injectable organic esters such as ethyl oleate, and suitablemixtures thereof.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound may be mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier, such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol and silicic acid; b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia; c) humectants such as glycerol; d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate; e) solutionretarding agents such as paraffin; f) absorption accelerators such asquaternary ammonium compounds; g) wetting agents such as cetyl alcohol,glycerol monostearate, and PEG caprylic/capric glycerides; h) absorbentssuch as kaolin and bentonite clay and i) lubricants such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate and mixtures thereof. In the case of capsules, tabletsand pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well-known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and may also be of acomposition such that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof.

Besides inert diluents, the oral compositions may also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring and perfuming agents.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active compound.

Compounds of the present invention can also be administered in the formof liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multi-lamellar hydrated liquid crystals which are dispersed inan aqueous medium. Any, physiologically acceptable and metabolizablelipid capable of forming liposomes can be used. The present compositionsin liposome form can contain, in addition to a compound of the presentinvention, stabilizers, preservatives, excipients and the like. Thepreferred lipids are natural and synthetic phospholipids andphosphatidyl cholines (lecithins) used separately or together.

Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq.

In another aspect, the invention is directed to a method of treating adisease or condition in a subject comprising administering to thesubject a therapeutically effective amount of a compound of any ofFormulas I-II or a pharmaceutically acceptable salt, ester, or prodrugthereof. The preferred route of administering to the subject is via oraldelivery.

Preferably, diseases or conditions treatable with the compounds of thepresent invention are related to the CNS. In a preferred embodiment, thedisease is schizophrenia.

However, it is within a skill in the art that the provided compounds maybe used to treat other diseases or conditions associated with diminishedglutathione levels and/or glutamate signaling, and/or oxidative stress,and/or impaired cystine-glutamate antiporter activity, glutamateneurotransmission, synaptic connection, and gene expression.

In general, the invention is not limited to treatment of any specificdisease or condition but encompasses the treatment of any disease orcondition whose mechanism may be affected by the compounds of thepresent invention.

In another aspect, the invention provides a method of treating drugcraving in a subject comprising administering to the subject atherapeutically effective amount of a compound of any of Formulas I-IIor a pharmaceutically acceptable salt, ester or prodrug thereof. Thepreferred route of administering to the subject is via oral delivery.

The invention further encompasses pharmaceutical compositions containinga compound of any of Formulas I-II or a pharmaceutically acceptablesalt, ester or prodrug thereof in combination with apharmaceutically-acceptable carrier.

Methods of formulating/manufacturing such pharmaceutical compositions(alternatively termed “medicaments”) for the treatment of a disease orcondition in a subject are also within the invention's scope.

For a clearer understanding of the invention, Examples are providedbelow. These are merely illustrations and are not to be understood aslimiting the scope of the invention in any way. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from thefollowing examples and foregoing description. Such modifications arealso intended to fall within the scope of the appended claims.

EXAMPLES Example 1—Synthesis Strategies

General Methods

All solvents used were commercially available and were used withoutfurther purification. Reactions were typically run using anhydroussolvents under an inert atmosphere of nitrogen. Compounds are namedusing ChemDraw 7, Reaxys or catalogue names if commercially available.

¹H and ¹³C NMR spectra were recorded at 400 MHz for proton and 100 MHzfor carbon-13 on a Varian 300 MercuryPlus station with an Oxford AS400Spectrometer equipped with a Varian 400 ATB PFG probe. All deuteratedsolvents contained typically 0.03% to 0.05% v/v tetramethylsilane, whichwas used as the reference signal (set at δ 0.00 for ¹H and ¹³C). For¹³C, the shifts were relative to the DMSO-d6 assignment of δδ 39.50.

Mass spectra were recorded on a Waters MS consisting of an Alliance 2795(LC) and Waters Micromass ZQ detector at 120° C. The mass spectrometerwas equipped with an electrospray ion source (ES) operated in a positiveor negative mode. The mass spectrometer was scanned between m/z 100-1000with a scan time of 0.3 s.

Elemental Analysis for C, H and N composition was performed using aCostech Instrument Elemental Combustion System ECS4010 with a heliumflow of 100 mL/min (14 psi), oxygen 20 mL/min (10 psi), air 25 psi andpurge of 50 mL/min (N42) or at the University of Alberta Analytical andInstrumentation Laboratory (N39).

Ultra-performance liquid chromatography (UPLC) analyses were performedon a Water 600 Controller system with a Waters 717 Plus Autosampler anda Waters 2996 Photodiode Array Detector. The column used was an AtlantisT3 d18 4.6×150 mm 3 μm and a gradient was applied, starting at 100% A(A: 0.1% H₃PO₄ in water) and ending at 30% (B: MeCN) over 10 min andthen increased to 95% B where it was maintained for 2 min. The columnwas then re-equilibrated to 100% A for the remainder of the 20 min. Thecolumn temperature was at ambient temperature with the flow rate of0.9-1.2 mL/min. The Diode Array Detector was scanned from 200-400 nm.

Thin layer chromatography (“TLC”) was performed on Alugram® (silica gel60 F₂₅₄; Alugram is a registered trademark of Macherey, Nagel & Co.) andultra violet light (“UV”) was typically used to visualize the spots.Additional visualization methods were also employed in some cases. Forexample, the TLC plate could also be developed with iodine (generated byadding approximately 1 g of I₂ to 10 g silica gel and thoroughlymixing), vanillin (generated by dissolving about 1 g vanillin in 100 mL10% H₂SO₄), ninhydrin (available commercially from Aldrich), or MagicStain (generated by thoroughly mixing 25 g (NH₄)₆Mo₇O₂₄.4H₂O, 5 g(NH₄)₂Ce(IV)(NO₃)₆ in 450 mL water and 50 mL concentrated H₂SO₄) tovisualize the compound. Medium pressure chromatography was performedwith a Biotage SP4® (Biotage is a registered trademark of Biotage AB)using SNAP™ silica gel cartridges or Teledyne Isco cartridges. Flashchromatography was preformed using typically 40-63 μm (230-400 mesh)silica gel from Silicycle following analogous techniques to thosedisclosed in Still et al. (Still, W. C.; Kahn, M.; and Mitra, M.,Journal of Organic Chemistry, 43: 2923-2925 (1978)). Typical solventsused for Biotage®, flash chromatography or thin layer chromatographywere mixtures of chloroform/methanol, dichloromethane/methanol, ethylacetate/methanol and hexanes/ethyl acetate.

Optical rotation was performed on a Perkin Elmer 241 Polarimeter at theUniversity of Alberta, Edmonton, Alberta Analytical and InstrumentationLab with a 10.002 cm path length.

Melting points were determined on an Electrothermal Digital MeltingPoint Apparatus (S.No 2345, Cat. No. IA8101) and are uncorrected.

The following abbreviations have been used:

-   aq. aqueous;-   DMSO dimethylsulfoxide;-   EtOAc ethyl acetate;-   HOAc acetic acid;-   MeOH methanol;-   NMM 4-methylmorpholine;-   ON overnight;-   r.t. room temperature;-   TFA trifluoroacetic acid; and-   THF tetrahydrofuran.

Starting materials used were available from commercial sources and usedas received.

Synthesis of N42:(2R)-2-Acetylamino-3-[(4R)-2-oxo-thiazolidine-4-carbonylsulfanyl]-propionicacid

The following procedure was performed based on the thioester formationconditions reported in Katritzky, A. R.; Tala, S. R.; Abo-Dya, N. E.;Ibrahim, T. S.; El-Feky, S. A.; Gyanda, K.; Pandya, K. M., J. Org.Chem., 76: 85-96 (2011).

(2R)-2-Acetylamino-3-[(4R)-2-oxo-thiazolidine-4-carbonylsulfanyl]-propionicacid (N42, Ref. 10-015-161)

To a solution of 1H-benzotriazole (19.4 g, 163 mmol) in THF (180 mL) wasadded thionyl chloride (2.96 mL, 40.8 mmol). The solution was cooled inan icebath and after 0.5 h, (4R)-2-oxo-thiazolidine-4-carboxylic acid(OTZ, 6.00 g, 40.8 mmol) was added. The solution warmed slowly toambient temperature over 1 h. After 1.25 h at room temperature, theresulting solid was removed by vacuum filtration. With 60 mL THF to washthe solid and flask, the filtrate was transferred to an ice coldsolution of (2R)-2-acetylamino-3-mercapto-propionic acid (NAC, 6.00 g,36.7 mmol) and NMM (4.04 mL, 36.7 mmol) in THF (30 mL). The ice bath wasremoved. After 30 min, the reaction appeared complete by ¹HNMR. To themixture was added TFA and the resulting solution was split in two andapplied to two 330 g silica gel Isco columns pre-wet with ethyl acetate.Biotage chromatography of the two columns (EtOAc to 20% methanol (0.5%AcOH)) generated 2.36 g (97.35% HPLC purity) of the title compound afterEtOAc trituration and drying in vacuo. Addition compound was obtained byconcentration the fast running UV active fractions and triturating withEtOAc. The solid was then dissolved in water, filtered and lyophilizedgenerating the title compound as a white lyophilizate. This material wascombined with a batch from the EtOAc mother liquor and was absorbed to25 g silica gel with ˜1:1 EtOAc-MeOH. Biotage purification (330 g Isco,EtOAc to 20% methanol (0.5% AcOH), pre-wet column with EtOAc) generated0.84 g (light yellow lyophilizate, 94.70% HPLC purity) and 3.74 g (whitelyophilizate, 96.88% HPLC purity) after dissolving in water, filteringand lyophilizing. Yield: 6.94 g (65%).

¹H NMR (400 MHz, DMSO-d₆) δ 13.01 (br.s, 1H), 8.95 (d, J=2.0 Hz, 1H),8.33 (d, J=8.2 Hz, 1H), 4.61-4.57 (m, 1H), 4.39-4.33 (m, 1H), 3.81 (dd,J=11.7, 9.0 Hz, 1H), 3.43 (dd, J=11.7, 2.3 Hz, 1H), 3.39 (dd, J=13.3,4.7 Hz, 1H), 3.08 (dd, J=13.7, 8.6 Hz, 1H), 1.84 (s, 3H); ¹³C NMR (100.6MHz, DMSO-d₆) 200.9, 173.5, 171.6, 169.4, 61.7, 51.2, 32.7, 30.0, 22.3ppm; MS (ES) m/z: 293 (M+H)⁺; HPLC: 96.88% (MaxPlot 220-400 nm);Elemental analysis for C₉H₁₂N₂O₅S₂: Calcd: C, 36.98%; H, 4.14%; N,9.58%. Found: C, 36.80%; H, 4.24%; N, 9.63%. [α]D²⁵−71.58 (c 1.0,water).

Synthesis of N50:(2R)-2-Acetylamino-3-(2-methoxy-benzoylsulfanyl)-propionic acid

The following procedure was performed based on the thioester formationconditions reported in Katritzky, A. R.; Tala, S. R.; Abo-Dya, N. E.;Ibrahim, T. S.; El-Feky, S. A.; Gyanda, K.; Pandya, K. M., J. Org.Chem., 76: 85-96 (2011).

(2R)-2-Acetylamino-3-(2-methoxy-benzoylsulfanyl)-propionic acid (N50,Ref. 10-015-179-7)

To a solution of 1H-benzotriazole (7.68 g, 64.4 mmol) in THF (60 mL) wasadded 2-methoxy-benzoyl chloride (4.36 mL, 29.3 mmol). After 15 min theresulting solution turned cloudy and the mixture was stirred for 1.75 hlonger. The resulting solid was removed by vacuum filtration with 15 mLTHF wash. The resulting filtrate, with 15 mL THF wash, was added topre-cooled solution of (2R)-2-acetylamino-3-mercapto-propionic acid(NAC, 3.83 g, 23.4 mmol) and NMM (2.57 mL, 23.4 mmol) in THF (30 mL).The ice bath was removed. After overnight a room temperature thereaction was not complete by ¹HNMR. More NMM (2.57 mL, 23.4 mmol) wasadded and the mixture was heated to 55° C. overnight. To the mixture wasadded TFA (4 mL) and the solution was concentrated in vacuo. The residuewas dissolved in MeOH and EtOAc and was absorbed to ˜80 g silica gel.Purification was accomplished by Biotage silica gel chromatography (330g ISCO column, 120 mL 1:2 EtOAc-hexanes then 120 mL 1:1 then EtOAc to20% MeOH (1% AcOH) gradient) followed by precipitation by dissolving ina warm MeCN/water 1:1 mixture (100 mL), removing the MeCN in vacuo, andcollecting the precipitate by suction filtration, with water wash, afterstanding overnight at ambient temperature. The title compound wasisolated as a white solid. Yield: 2.29 g (33%).

Mp 179-181° C. ¹H NMR (399.7 MHz, DMSO-d₆) δ 12.89 (br.s, 1H), 8.31 (d,J=8.2 Hz, 1H), 7.65 (dd, J=7.8, 1.6 Hz, 1H), 7.59-7.54 (m, 1H), 7.18 (d,J=8.6 Hz, 1H), 7.06-7.02 (m, 1H), 4.42-4.36 (m, 1H), 3.85 (s, 3H), 3.47(dd, J=13.7, 5.1 Hz, 1H), 3.11 (dd, J=13.7, 9.0 Hz, 1H), 1.82 (s, 3H);¹³C NMR (100.5 MHz, DMSO-d₆) 189.4, 171.8, 169.3, 157.4, 134.3, 129.0,125.9, 120.4, 113.8, 55.9, 51.4, 30.3, 22.3 ppm; MS (ES) m/z: 298(M+H)⁺; HPLC: 97.72% (MaxPlot 220-400 nm); Elemental analysis forC₁₃H₁₅NO₅S: Calcd: C, 52.51%; H, 5.09%; N, 4.71%; S, 10.78%. Found: C,52.58%; H, 5.07%; N, 4.82%; S, 10.55%. [α]D²⁵−21.49 (c 1.014, DMSO).

Synthesis of N51:(2R)-2-Acetylamino-3-(4-methyl-benzoylsulfanyl)-propionic acid(Pro-2023)

The following procedure was performed based on the thioester formationconditions reported in Katritzky, A. R.; Tala, S. R.; Abo-Dya, N. E.;Ibrahim, T. S.; El-Feky, S. A.; Gyanda, K.; Pandya, K. M., J. Org.Chem., 76: 85-96 (2011).

(2R)-2-Acetylamino-3-(4-methyl-benzoylsulfanyl)-propionic acid (N51,Ref. 10-015-177-7)

To a solution of 1H-benzotriazole (9.91 g, 83.2 mmol) in THF (80 mL) wasadded 4-methyl-benzoyl chloride (5.00 mL, 37.8 mmol). After 2 h, theresulting solid was removed by vacuum filtration with 25 mL THF wash.The resulting filtrate, with 25 mL THF wash, was added to pre-cooledsolution of (2R)-2-acetylamino-3-mercapto-propionic acid (NAC, 5.55 g,34.0 mmol) and NMM (3.74 mL, 34.0 mmol) in THF (50 mL). The ice bath wasremoved. After overnight a room temperature the reaction was notcomplete by ¹HNMR. The mixture was heated to 55° C. for 1 h. To themixture was added TFA (3.6 mL) and water (100 mL). The solution wasconcentrated in vacuo to remove most of the THF. More water (80 mL) anddichloromethane (180 mL) were added and the layers separated. Theaqueous layer was extracted with dichloromethane. The organic layerswere combined, dried over Na₂SO₄, filtered, ethyl acetate added (100 mL)and concentrated in vacuo to remove dichloromethane. The remaining ethylacetate solution was place briefly in the freezer to startcrystallization then left out overnight at ambient temperature. Thesolid obtained was collected by vacuum filtration generating 3.28 gafter drying and the filtrate, with some methanol added, was absorbed to85 g silica gel. Biotage column chromatography (330 g ISCO,hexanes-ethyl acetate 1:1 (0.5 CV) then EtOAc to 20% MeOH (1% AcOH)gradient) yielded 2.62 g of material. The solid from EtOAc precipitationand the material off the column were combined and dissolved in warmMeCN/water 1:1 mixture. The MeCN was removed in vacuo, and theprecipitate that formed after leaving at ambient temperature overnightwas collected by suction filtration. The title compound was isolated asa white solid. Yield: 4.81 g (50%).

Mp 182-184° C. ¹H NMR (399.7 MHz, DMSO-d₆) □ 12.98 (br.s, 1H), 8.38 (d,J=7.8 Hz, 1H), 7.82 (d, J=8.0 Hz, 2H), 7.37 (d, J=8.0 Hz, 2H), 4.47-4.42(m, 1H), 3.55 (dd, J=13.7, 5.1 Hz, 1H), 3.23 (dd, J=13.7, 8.6 Hz, 1H),2.89 (s, 3H), 1.84 (s, 3H); ¹³C NMR (100.5 MHz, DMSO-d₆) 189.9, 171.7,169.3, 144.6, 133.6, 129.6, 127.0, 51.4, 29.9, 22.3, 21.2 ppm; MS (ES)m/z: 282 (M+H)⁺; HPLC: 94.77% (MaxPlot 220-400 nm); Elemental analysisfor C₁₃H₁₅NO₄S: Calcd: C, 55.50%; H, 5.37%; N, 4.98%; S, 11.40%. Found:C, 55.46%; H, 5.33%; N, 5.08%; S, 11.66%. [α]D²⁵−23.74 (c 1.0, DMSO).

Synthesis of N53:(2R)-2-Acetylamino-3-benzyloxycarbonylsulfanyl-propionic acid

The following procedure was performed based on the thiocarbonateformation conditions reported in Crankshaw, D. L.; Berkely, L. I.;Cohen, J. F.; Shirota, F. N.; Nagasawa, H. T., Journal of Biochemicaland Molecular Toxicology, 16: 235-244 (2002).

(2R)-2-Acetylamino-3-benzyloxycarbonylsulfanyl-propionic acid (N53, Ref.10-015-183-7)

To a solution of 2R)-2-acetylamino-3-mercapto-propionic acid (NAC, 4.02g, 24.6 mmol) in water (30 mL) was added sodium carbonate (2.64 g, 24.9mmol) followed by THF (30 mL). Then benzyl chloroformate (7.73 mL, 54.1mmol) was added. After 1 h, more sodium carbonate was added to adjust pHto ˜8. After additional 0.5 h, partially concentrate in vacuo. Extractaqueous with EtOAc (3×) then acidify aqueous with 2 N HCl to pH˜3. Addether and separate layers. Extract aqueous with EtOAc (2×) and combineorganic layers, dry over Na₂SO₄, filter and concentrate in vacuo.Purification was accomplished by Biotage column chromatography (330 gISCO, 1:1 hex/EtOAc (120 mL) then EtOAc to 20% MeOH (1% AcOH) gradient,sample loaded in EtOAc with some hexanes) generated the title compoundas a white solid. Yield: 2.18 g (30%).

Mp 146-148° C. ¹H NMR (399.7 MHz, DMSO-d₆) δ 12.98 (br.s, 1H), 8.33 (d,J=8.2 Hz, 1H), 7.40-7.32 (m, 5H), 5.27-5.20 (m, 2H), 4.44-4.38 (m, 1H),3.35-3.30 (dd, partially masked by H₂O, 1H), 3.05 (dd, J=14.0, 8.6 Hz,1H), 1.81 (s, 3H); ¹³C NMR (100.5 MHz, DMSO-d₆) 171.6, 169.6, 169.4,135.2, 128.5₄, 128.4₇, 128.3₈, 68.9, 51.5, 32.1, 22.3 ppm; MS (ES) m/z:298 (M+H)⁺; HPLC: 98.22% (MaxPlot 220-400 nm); Elemental analysis forC₁₃H₁₅NO₅S: Calcd: C, 52.51%; H, 5.09%; N, 4.71%; S, 10.78%. Found: C,pending %; H, pending %; N, pending %; S, pending %. [α]D²⁵ pending(c,).

Specific Methods

¹H-NMR spectra were obtained on a Varian Mercury 300-MHz NMR. Purity (%)was determined with a Waters Alliance 2695 HPLC (Waters Symmetry C18,4.6×75 mm, 3.5 μm) with a 2996 diode array detector from 210-400 nm.

Synthesis of Ethyl(2R)-2-acetamido-3-(4-methylbenzoylsulfanyl)propanoate (Pro-4051)

2-Acetylamino-3-(4-methyl-benzoylsulfanyl)-propionic acid

To a solution of benzotriazole (9.91 g, 83.2 mmol) in tetrahydrofuran(80 mL) was added 4-methylbenzoyl chloride (5.0 mL, 37.8 mmol) (5 g,95%). After 2 hours, the resulting slurry was filtered and the solid wasrinsed with tetrahydrofuran (25 mL). The filtrates were combined andadded to a solution of N-acetyl-L-cysteine (5.55 g, 34.0 mmol) andN-methylmorpholine (3.74 mL, 34.0 mmol) in tetrahydrofuran (50 mL) at 0°C. The resulting mixture was allowed to warm to ambient temperature over16 hours. Aqueous hydrochloric acid (1 M, 100 mL) was added to themixture and the resulting mixture was concentrated at reduced pressureto a volume of approximately 125 mL. Additional aqueous hydrochloricacid (80 mL) was added and the mixture was extracted withdichloromethane (180 mL then 80 mL). The combined organic extracts werecombined, dried over sodium sulfate, filtered and concentrated atreduced pressure. Ethyl acetate (100 mL) was added and the resultingslurry was stirred for 2 hours. The resulting slurry was filtered anddried to give a white solid (5.7 g, 60%). ¹H NMR (300 MHz, DMSO) δ=12.94(s, 1H), 8.34 (d, J=7.9 Hz, 1H), 7.86-7.74 (m, 2H), 7.35 (d, J=8.5 Hz,2H), 4.43 (dt, J=5.0, 8.4 Hz, 1H), 3.53 (dd, J=5.0, 13.8 Hz, 1H), 3.21(dd, J=8.5, 13.8 Hz, 1H), 2.37 (s, 3H), 1.83 (s, 3H). MS (ESI) m/z 282(M+1)⁺.

Ethyl (2R)-2-acetamido-3-(4-methylbenzoylsulfanyl)propanoate

To a solution of 2-acetylamino-3-(4-methyl-benzoylsulfanyl)-propionicacid (1.1 g, 3.91 mmol) and triethylamine (0.68 mL, 4.89 mmol)N,N-dimethylformamide (5 mL) was added iodoethane (0.39 mL, 4.89 mmol)and the resulting solution was stirred at ambient temperature for 18hours. The reaction mixture was added slowly to water (50 mL) with rapidstirring. After stirring for 2 hours, the resulting slurry was filteredand the solid was rinsed with water and dried under vacuum to give theproduct as a white solid (780 mg, 60%). ¹H NMR (300 MHz, DMSO) δ=8.47(d, J=7.9 Hz, 1H), 7.81 (s, 1H), 7.80-7.78 (m, 1H), 7.35 (d, J=7.6 Hz,2H), 4.46 (dt, J=5.3, 8.1 Hz, 1H), 4.09 (q, J=6.8 Hz, 2H), 3.50 (dd,J=5.4, 13.6 Hz, 1H), 3.24 (dd, J=8.2, 13.8 Hz, 1H), 2.37 (s, 3H), 1.83(s, 3H), 1.16 (t, J=7.0 Hz, 3H). MS (ESI) m/z 310 (M+1)⁺.

Synthesis of (2R)-2-acetamido-3-(4-methylbenzoylsulfanyl)propanamide(Pro-4051A)

To a solution of 2-acetylamino-3-(4-methyl-benzoylsulfanyl)-propionicacid (500 mg, 1.78 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (686 mg, 3.58 mmol), hydroxybenzotriazole hydrate (483 mg,3.58 mmol) and triethylamine (0.50 mL, 3.58 mmol) inN,N-dimethylformamide (5 mL) was added ammonium chloride (191 mg, 3.58mmol) and the resulting slurry was stirred at ambient temperature for 18hours after which time a solution formed. The mixture was diluted withethyl acetate (100 mL) and wash successively with hydrochloric acid (0.1M, 50 mL), saturated aqueous sodium hydrogen carbonate (50 mL). theorganic layer was dried over magnesium sulfate, filtered andconcentrated at reduced pressure to give an oil. This crude product waspurified with silica gel (12 g) chromatography eluting with 50 to 100%ethyl acetate in hexane. The purified fractions were combined andconcentrated at reduced pressure to give the product as a white solid(55 mg, 11%). ¹H NMR (300 MHz, DMSO) δ=8.16 (d, J=8.5 Hz, 1H), 7.79 (d,J=8.2 Hz, 2H), 7.47 (br. s., 1H), 7.35 (d, J=8.2 Hz, 2H), 7.19 (br. s.,1H), 4.43 (dt, J=5.4, 8.3 Hz, 1H), 3.42 (dd, J=5.4, 13.3 Hz, 1H), 3.16(dd, J=8.4, 13.3 Hz, 1H), 2.32 (s, 3H), 1.85 (s, 3H). MS (ESI) m/z 281(M+1)⁺.

Synthesis of(2R)-2-Acetamido-3-[(2-phenylpropan-2-yl)sulfanyl]propanamide (Pro-4006)

(2R)-2-Amino-3-(1-methyl-1-phenyl-ethylsulfanyl)-propionic acidhydrochloride

To a solution of L-cysteine hydrochloride (2 g, 12.7 mmol) in aqueoushydrochloric acid (2M, 30 mL) was added 2-phenyl-propan-2-ol (1.78 mL,12.7 mmol) and the resulting mixture was heated in an oil bath at 65-75°C. for 18 hours. The reaction mixture was cooled and filtered and rinsedwith aqueous hydrochloric acid (1M, 10 mL) to give a white solid (2.2 g,63%). ¹H NMR (300 MHz, DMSO) δ=8.62 (br. s, 3H), 7.55-7.43 (m, 2H),7.37-7.19 (m, 3H), 7.23-7.17 (m, 1H), 3.80 (m, 1H), 2.70-2.64 (m, 2H),1.64 (s, 6H).

(2R)-2-Acetylamino-3-(1-methyl-1-phenyl-ethylsulfanyl)-propionic acid

To a mixture of(2R)-2-amino-3-(1-methyl-1-phenyl-ethylsulfanyl)-propionic acidhydrochloride (1.0 g, 3.63 mmol) in water (5 mL) and 1,4-dioxane (5 mL)at 0° C. was added aqueous sodium hydroxide (2M) to pH 10, then aceticanhydride (0.34 mL, 3.63 mmol) was added dropwise. The reaction mixturewas warmed to ambient temperature over 16 hours. The reaction mixturewas acidified to pH 2 with aqueous hydrochloric acid (1 M) and extractedwith ethyl acetate (60 mL×2). The combined organic layers were driedover magnesium sulfate, filtered and concentrated at reduced pressure togive the crude product which was used in the next step without furtherpurification.

(2R)-2-Acetamido-3-[(2-phenylpropan-2-yl)sulfanyl]propanamide

To a solution of(2R)-2-acetylamino-3-(1-methyl-1-phenyl-ethylsulfanyl)-propionic acid(400 mg, 1.42 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (545 mg, 2.84 mmol), hydroxybenzotriazole hydrate (384 mg,2.84 mmol) and triethylamine (0.40 mL, 2.84 mmol) inN,N-dimethylformamide (2 mL) was added ammonium chloride (152 mg, 2.84mmol) and the resulting slurry was stirred at ambient temperature for 18hours after which time a solution formed. The mixture was diluted withethyl acetate (100 mL) and wash successively with hydrochloric acid (0.1M, 50 mL), saturated aqueous sodium hydrogen carbonate (50 mL). Theorganic layer was dried over magnesium sulfate, filtered andconcentrated at reduced pressure to give an oil. This crude product waspurified with silica gel (12 g) chromatography eluting with 50 to 100%ethyl acetate in hexane. The purified fractions were combined andconcentrated at reduced pressure to give the product as a white solid(145 mg, 36%). ¹H NMR (300 MHz, DMSO) δ=7.92 (d, J=8.2 Hz, 1H),7.52-7.46 (m, 2H), 7.37-7.28 (m, 3H), 7.23-7.17 (m, 1H), 7.04 (s, 1H),4.21 (dt, J=6.0, 8.1 Hz, 1H), 2.47-2.32 (m, 2H), 1.79 (s, 3H), 1.62 (d,J=4.1 Hz, 6H). MS (ESI) m/z 281 (M+1)⁺.

Synthesis of Ethyl(2R)-2-acetamido-3-(2-oxo-1,3-thiazolidine-4-carbonylsulfanyl)propanoate (Pro-4047)

To a solution of L-2-oxothiazolidine-4-carboxylic acid (6.0 g, 40.8mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(9.03 g, 47.1 mmol) and hydroxybenzotriazole hydrate (6.4 g, 47.1 mmol)in N,N-dimethylformamide (30 mL) was added2-acetylamino-3-mercapto-propionic acid ethyl ester (6.0 g, 31.4 mmol)and the resulting slurry was stirred at ambient temperature for 72 hoursduring which time a solution formed. The mixture was diluted withaqueous hydrochloric acid (0.2 M, 300 mL) and extracted with ethylacetate (4×200 mL). The combined organic layers were washed withsaturated aqueous sodium hydrogen carbonate (150 mL). The organic layerwas dried over magnesium sulfate, filtered and concentrated at reducedpressure. This crude product was purified with silica gel (120 g)chromatography eluting with 30 to 100% ethyl acetate in hexane. Thepurified fractions were combined and concentrated at reduced pressure.The resulting solid was triturated with ethyl acetate/hexanes (1:1, 100mL), filtered and dried to give the product as a white solid (3.1 g,31%). ¹H NMR (300 MHz, CDCl₃) δ=6.33-6.20 (m, 2H), 4.87 (ddd, 5.9, 7.5Hz, 1H), 4.43 (ddd, J=1.8, 3.5, 8.5 Hz, 1H), 4.22 (dq, J=2.3, 7.1 Hz,2H), 3.79 (dd, J=8.5, 11.4 Hz, 1H), 3.62-3.51 (m, 2H), 3.40-3.31 (m,1H), 2.05-2.02 (m, 3H), 1.60 (s, 2H), 1.31 (t, J=7.2 Hz, 3H). MS (ESI)m/z 321 (M+1)⁺.

Synthesis of (6R)-1-benzyl-6-(sulfanylmethyl)piperazine-2,5-dione

(2R)-2-Benzylamino-3-tritylsulfanyl-propionic acid methyl ester

To a solution of (2R)-2-Amino-3-tritylsulfanyl-propionic acid methylester (41 g, 109 mmol) in methanol (570 mL) was added benzaldehyde (13.3mL, 131 mmol). After 1.5 hours, the reaction mixture was cooled in anice bath and sodium borohydride (8.25 g, 218 mmol) was added in portionsover 20 minutes. One addition was complete, the ice bath was removed andthe reaction was allowed to warm to ambient temperature over one hour.The resulting mixture was then concentrated under reduced pressure. Theresulting oil was diluted with water (300 mL) and ethyl acetate (500 mL)and the layers were separated. The organic layer was washed again withwater (200 mL) and the organic layer was dried over magnesium sulfate,filtered and concentrated at reduced pressure. The crude product waspurified with silica gel (330 g) chromatography eluting with 0 to 20%ethyl acetate in hexane. The purified fractions were combined andconcentrated at reduced pressure to give the product as a colorless oil(30.5 g, 60%). ¹H NMR (300 MHz, CDCl₃) δ=7.44-7.37 (m, 6H), 7.31-7.17(m, 14H), 3.69-3.62 (m, 4H), 3.60-3.51 (m, 1H), 3.14-3.07 (m, 1H), 2.50(d, J=6.4 Hz, 2H).

(6R)-1-benzyl-6-{[(triphenylmethyl)sulfanyl]methyl}piperazine-2,5-dione

To a solution of (2R)-2-benzylamino-3-tritylsulfanyl-propionic acidmethyl ester (30.2 g, 64.6 mmol) and diisopropylethylamine (12.4 mL, 71mmol) in dichloromethane (400 mL) at 0° C. was added bromoacetyl bromide(6.2 mL, 71 mmol) in dichloromethane (30 mL) dropwise via an additionfunnel. The reaction mixture was allowed to warm to ambient temperatureover 2 hours. The reaction was diluted with dichloromethane (300 mL) andextracted with aqueous hydrochloric acid (0.5 N, 400 mL). The aqueouslayer was extracted with dichloromethane (100 mL) and the combinedorganic extracts were dried over magnesium sulfate, filtered andconcentrated at reduced pressure. To this crude product was added asolution of ammonia in methanol (7 M, 200 mL) and the resulting solutionwas stirred at ambient temperature over 64 hours. The reaction mixturewas concentrated at reduced pressure. The resulting mixture wastriturated vigorously with aqueous hydrochloric acid (200 mL) for 3hours and filtered. The solid was dissolved in dichloromethane andpurified with silica gel (330 g) chromatography eluting with 0 to 70%ethyl acetate in hexane. The purified fractions were combined andconcentrated at reduced pressure to give the product as a white solid(14.1 g, 44% over two steps). ¹H NMR (300 MHz, CDCl₃) δ=7.42-7.37 (m,6H), 7.33-7.21 (m, 12H), 7.02 (dd, J=2.8, 6.6 Hz, 2H), 6.64 (d, J=2.6Hz, 1H), 5.26 (d, J=15.0 Hz, 1H), 4.27 (d, J=17.3 Hz, 1H), 3.98-3.83 (m,2H), 3.20 (d, J=15.0 Hz, 1H), 2.94 (dd, J=3.7, 12.5 Hz, 1H), 2.55 (dd,J=5.0, 12.6 Hz, 1H). MS (ESI) m/z 493 (M+1)⁺.

(6R)-1-benzyl-6-(sulfanylmethyl)piperazine-2,5-dione

To a solution of(6R)-1-benzyl-6-{[(triphenylmethyl)sulfanyl]methyl}piperazine-2,5-dione(13 g, 26.4 mmol) in trifluoroacetic acid (100 mL) and dichloromethane(200 mL) at 0° C. was added triisopropylsilane (21.6 mL, 106 mmol)dropwise. After addition was complete, the reaction mixture was allowedto warm to ambient temperature over 2 hours. Heptane (100 mL) was addedand the mixture was concentrated at reduced pressure. The crude productwas purified with silica gel (120 g) chromatography eluting with 0 to100% ethyl acetate in hexane. The purified fractions were combined andconcentrated at reduced pressure. The solid was triturated with ethylacetate (50 mL) to give the product as an off-white solid (6.0 g, 90%).¹H NMR (300 MHz, CDCl₃) δ=7.38-7.26 (m, 5H), 6.63 (br. s., 1H), 5.20 (d,J=15.0 Hz, 1H), 4.43 (d, J=17.3 Hz, 1H), 4.16-4.05 (m, 3H), 4.01 (d,J=2.9 Hz, 1H), 3.12 (ddd, J=2.3, 10.2, 14.7 Hz, 1H), 2.96 (ddd, J=4.1,8.4, 14.8 Hz, 1H), 1.65 (s, 1H), 1.41 (dd, J=8.4, 10.1 Hz, 1H). MS (ESI)m/z 251 (M+1)⁻.

Synthesis of(6R)-6-[(benzoylsulfanyl)methyl]-1-benzylpiperazine-2,5-dione (Pro-4011)

To a stirring solution of(6R)-1-benzyl-6-(sulfanylmethyl)piperazine-2,5-dione (200 mg, 0.79 mmol)in pyridine (3 mL) at 0° C. was added benzoyl chloride (0.23 mL, 1.98mmol) dropwise and when addition was complete, the reaction mixture waswarmed to ambient temperature and stirred for 24 hours. The reaction wasconcentrated at reduced pressure, diluted with dichloromethane (100 mL)was washed with saturated aqueous sodium hydrogen carbonate (30 mL) thenaqueous hydrochloric acid (0.1N, 40 mL). The organic layer was driedover magnesium sulfate, filtered and concentrated at reduced pressure.The crude product was purified with silica gel (20 g) chromatographyeluting with 20 to 100% ethyl acetate in hexane. The purified fractionswere combined and concentrated at reduced pressure. The solid wastriturated with ethyl acetate/hexanes (1:1, 10 mL) to give the productas a white solid (77 mg, 27%). ¹H NMR (300 MHz, DMSO) δ=8.37 (d, J=3.5Hz, 1H), 7.92 (d, J=7.8 Hz, 2H), 7.74-7.67 (m, 1H), 7.60-7.53 (m, 2H),7.37-7.24 (m, 5H), 5.06 (d, J=15.0 Hz, 1H), 4.21-4.10 (m, 2H), 4.04 (dd,5.7 Hz, 1H), 3.81-3.71 (m, 2H), 3.55 (dd, J=3.8, 14.1 Hz, 1H), 3.40-3.34(m, 1H), 3.32-3.25 (m, 1H). MS (ESI) m/z 355 (M+1)⁺.

Example 2—In Vitro Studies

¹⁴C Uptake by Compounds of the Invention

The goal of these experiments was to determine ¹⁴C-cystine uptake.

The experiments were conducted as follows.

The screening of compounds was performed using an in vitro culturesystem of human glial cells from brain astrocytoma (1321N1). Cells wereplated on 24 well plates coated with poly-D-lysine and laminin and grownin a balanced salt solution supplemented with 5% heat inactivated horseserum, 5% fetal bovine serum, 2 mM glutamine and glucose (total 21 mM).Cultures were maintained in humidified 5% CO₂ incubators at 37° C. for3-4 days before experiments were performed, at this time the cultureshas formed a single confluent layer. For experiments, cultures werewashed 3 times into a Na-free HEPES and HCO₃ ⁻ buffered balanced saltsolution. After 1 hour, the test compounds are added. Following a threehour incubation, ¹⁴C-cystine (0.025 mCi/mL) was then added for 20minutes. Following the ¹⁴C-cystine exposure, cultures were washed 3times with ice cold HEPES buffered saline solution and dissolved in 250μl sodium dodecyl sulfate (0.1%). An aliquot (200 μl) was removed andadded to scintillation fluid for counting. Values were normalized to¹⁴C-cystine uptake in untreated controls on the same experimental plate.

³H-Glutamate Release by Compounds of the Invention

This assay also uses the cell culture system of human glial cells frombrain astrocytoma (1321N1) described above. Initially, cells are washedwith HBBSS, and ³H-glutamate is added (PerkinElmer: 1 mCi/mL stocksolution is diluted (30 μL+500 μL HBBSS) and 10 μL of diluted radiolabelis added to each well). Following a 1 hour incubation to load the cellswith the labeled glutamate, the cells are washed again with HBBSS, andthe drug is added. At 30, 90, and 180 minutes, 50 μL of extracellularmedia is sampled from each well and is measured using a Beckman LS 6500scintillation counter.

Results of the ¹⁴C-cystine uptake and ³H-glutamate release by astrocytestreated with differing concentrations of Pro-2022, Pro-2023, Pro-4006,Pro-4011, Pro-4047, Pro-4051 and Pro-4051a, are presented in FIGS. 1, 2and 5-9, respectively, as a percentage of the untreated control. Only¹⁴C-cystine uptake data was obtained for Pro-2024 and Pro-3010. The mosteffective concentration of Pro 2022 for radiolabeled cystine uptake was300 μM which surprisingly shows an increase of about 50% over theuntreated control. This data suggest interaction with the target (Systemxc-) albeit in an unexpected manner. The most effective concentration ofPro-2022 for glutamate release was 100 μM at 3 hours incubation whichproduced an approximately 60% increase. The most effective concentrationof Pro-2023 for radiolabeled cystine uptake was 1000 μM which producedan approximately 45% decrease in ¹⁴C-cystine uptake demonstratinginhibition of ¹⁴C-cystine uptake by a process not limited to analternate substrate, a cystine-glutamate antiporter inhibitor, and aneffective prodrug. The most effective concentration of Pro-2023 forglutamate release was 300 μM which produced an approximately 135%increase at 3 hours, which is evidence that the ¹⁴C-cystine uptake isbeing inhibited by direct competition from Pro-2023. The most effectiveconcentration of Pro-2024 for radiolabeled cystine uptake was 100 μMwhich surprisingly showed an increase. 300 and 1000 μM concentrationsproduced inhibition of radiolabeled cystine uptake of approximately 30%and 35%, respectively. The most effective concentration of Pro-3010 was1000 μM which produced an approximately 75% decrease in radiolabeledcystine uptake. The most effective concentration of Pro-4006 was 30 μMwhich produced an approximately 45% decrease in ¹⁴C-cystine uptake witha corresponding 35% increase in ³H-glutamate release at 30 minutes.Based on previous solubility studies with related analogues, it ispostulated that the 1000 μM dose is likely out of solution; nonetheless,we have included this high concentration of Pro-4006 in the current dataset. The most effective concentration of Pro-4011 was 100 μM whichsurprisingly produced an approximately 75% increase in ¹⁴C-cystineuptake with a corresponding 75% increase in ³H-glutamate release at 3hours. The 300 and 1000 μM data points may be aberrant due to theappearance of precipitates in the assay media suggesting the compoundwas not in solution. Pro-4047 produced an approximate 20-60% decrease in¹⁴C-cystine uptake demonstrating a dose-dependent decrease. The mosteffective concentration of Pro 4047 for glutamate release was 100 μMwhich produced an approximately 40% increase at 3 hours. The mosteffective concentration of Pro-4051 was 100 μM which produced anapproximately 28% decrease in ¹⁴C-cystine uptake. This graph representsan average of three experiments run. Pro-4051 is not in solution above316 μM according to solubility data. The most effective concentration ofPro-4051 for ³H-glutamate release was 300 μM which produced anapproximately 100% increase at 3 hours. Pro-4051a did not produce asubstantial inhibition of ¹⁴C-cystine uptake. This is likely because the3 hour incubation does not fall within the window of efficacy for thiscompound. The most effective concentration of Pro-4051a for ³H-glutamaterelease was 1000 μM which produced an approximately 180% increase at 30minutes.

In Vitro Thiol Experiments

To further resolve by what process the inhibition of ¹⁴C-cystine uptakeis occurring intracellular cysteine levels were determined.

The experiment was conducted as follows.

Mixed cortical cell cultures containing glial and neuronal cells wereprepared from fetal (15-16 day gestation) mice as previously described.(Lobner D, “Comparison of the LDH and MTT assays for quantifying celldeath: validity for neuronal apoptosis?,” J. Neurosci. Methods, 96(2):147-152 (Mar. 15, 2000)). Dissociated cortical cells were plated on24-well plates coated with poly-D-lysine and laminin in Eagles′ MinimalEssential Medium (MEM, Earle's salts, supplied glutamine-free)supplemented with 5% heat-inactivated horse serum, 5% fetal bovineserum, 2 mM glutamine and glucose (total 21 mM). Cultures weremaintained in humidified 5% CO₂ incubators at 37° C. Mice were handledin accordance with a protocol approved by our institutional animal carecommittee and in compliance with the Public Health service Policy onHumane Care and Use of Laboratory Animals.

Mixed cortical cell cultures, 14 days in vitro (this allows for aconfluent layer of astrocytes to form and the neurons to generate acomplex network of axons and dendrites), were washed into bicarbonatebuffered salt solution. After 1 hour, 3, 10, 30, 100 μM of Pro-4047 orPro-4051 was added and the cells were incubated for 30 or 90 minutes,after which the cells were thoroughly washed and the collected in 250 μLof aqueous mobile phase (50 mM citric acid, 10 mM octanesulfonic acid,pH 2.8) was added, after 10 minutes at 37° C. the cells were scrapedfrom the plates and transferred to 1.5 mL tubes for analysis.

Samples were then sonicated with a Fisher Scientific 60 SonicDismembrator. One fraction of this homogenate was analyzed with thePierce BCA (bicinchoninic acid) method to determine proteinconcentration. The other fraction was filtered using 3 K molecularweight cutoff, polyethersulfone centrifugal protein filters, andanalyzed for thiol content using HPLC (ALF-115 column, 150×1.0 mm, 3 μmC18 [Pro-2023 analysis utilized a Phenomenex Kinetex 2.6 μM, C18, 100A,150×2.1 mm and Pro-4047 analysis utilized a Phenomenex Kinetex X-B, C18,100 A, 2.6 μm, 150×4.4 mm]; mobile phase: 50 mM citric acid, 10 mMoctanesulfonic acid, 2% acetonitrile, pH 2.8, 50 μL/min flow rate[Pro-2023 analysis utilized a 100 μL/min flow rate and Pro-4047 analysisutilized 1% acetonitrile and 0.4 mL/min flow rate] with electrochemicaldetection (Decade II, Au working electrode, Flex Cell HyREF, 0.55V,Antec Leyden, Netherlands [Pro-4047 analysis utilized a Magic Diamondworking electrode and 1.8V]).

When the intracellular thiol assay was initiated, all data wasnormalized to protein concentration. However, the protein method did notproduce consistent results in control samples, so that method ofnormalization was abandoned. The data presented herein represents theraw cysteine concentrations in the collected samples. Resultsdemonstrate that administration of Pro-2023 at 100 μM increasesintracellular cysteine concentration to 0.16 μM at 30 minutes(approximately 1.6 times that of the control; FIG. 10). Pro-4006 at 10μM increases intracellular cysteine concentration to 0.07 μM at 30minutes (approximately 3.5 times that of the control; FIG. 11). Pro-4011at 10 μM increases intracellular cysteine concentration to 0.138 μM at30 minutes (approximately 1.16 times that of the control; FIG. 12).Pro-4047 at 300 μM increases intracellular cysteine concentration to0.74 μM at 90 minutes (approximately 4.9 times that of the control; FIG.13). FIG. 14 demonstrates that administration of Pro-4051 at 100 μMincreases intracellular cysteine concentration to 0.36 μM at 90 minutes(approximately 4 times that of the control). Finally, FIG. 15demonstrates that administration of Pro-4051a at 100 μM increasesintracellular cysteine concentration to 0.21 μM at 30 minutes(approximately 3 times that of the control). These results demonstratethat Pro-2023, Pro-4006, Pro-4011, Pro-4047, Pro-4051 and Pro-4051a wereeffectively cleaved, yielding an increase in intracellular cysteine.Thus, based on the 3 in vitro experiments it is apparent that Pro-2023,Pro-4006, Pro-4011, Pro-4047, Pro-4051 and Pro-4051a behave as effectivecysteine prodrugs.

Example 3—In Vivo Studies

Prepulse Inhibition Experiment

The goal of this experiment was to demonstrate the efficacy of the testcompounds in a predictive animal model of schizophrenia.

The experiment was conducted as follows.

Rats were placed on a platform in a sound attenuating chamber(10.875″×14″×19.5″; Hamilton Kinder, CA) that rested on a motion sensingplate. During all sessions, the background noise was held constant at 60dB. A matching session was conducted to determine the magnitude of theaverage startle response for each rat. This session consisted of a fiveminute habituation period followed by 20 trials; 17 trials involved thepresentation of a single auditory stimulus (pulse stimulus; 50 dB abovethe background noise) and three trials in which a pre-pulse stimulus (12db above background) was presented 100 ms before the pulse auditorystimulus. Rats were then assigned into the various treatment groups sothat the magnitude of the startle response was equivalent across allgroups. Two days later, a testing session was conducted to assesssensorimotor gating. One hour prior to testing, rats received a prodrug(0-100 mg/kg, P.O.) and 55 minutes later acute MK-801 maleate (0.1mg/kg, SC). The testing session consisted of a five minute habituationperiod, after which rats received 58 discrete trials; 26 trials duringwhich the pulse stimulus (50 db above background) was presented alone,eight trials each in which the pulse stimulus was preceded by a prepulsestimulus (5, 10, or 15 db above background) and eight background trialswith no pulse (No stimulus; background noise only). The first six pulsealone trials were not included in the average startle stimulus toachieve a relatively stable level of startle reactivity. All startleresponses were normalized to vehicle control, and the percent ofprepulse inhibition was determined as 100-(average prepulse startleresponse/average startle stimulus alone)*100.

FIG. 16 depicts the average prepulse inhibition for Pro-2023. As the bargraph demonstrates, at 10 mg/kg concentration, % prepulse inhibition wasabout 30% as opposed to about 16% for the control group that receivedboth MK-801 and vehicle. Consistent with similar dose-response curves inthis assay (including clozapine), the compound produces an invertedU-shaped dose response at higher concentrations. These data, which areconsistent to the effects of clinically-used neuroleptics, suggestantipsychotic-like activity in rodent model of schizophrenia.

FIG. 17 depicts the average prepulse inhibition for Pro-4047. As the bargraph demonstrates, at 3 mg/kg concentration, % prepulse inhibition wasabout 23% as opposed to about 16% for the control group that receivedboth MK-801 and vehicle. The general lack of efficacy in this model,despite activity in the primary cell-based assays, is likely due toeither pharmacokinetic parameters that are such that the therapeuticwindow was missed with the single time-point tested. Additionally, thiscould be due to the dose-range tested where higher doses (e.g. 30 or 60mg/kg) could have produced an effect.

FIG. 18 demonstrate the results of the experiment for Pro-4051. As thebar demonstrates, at 10 mg/kg concentration, % prepulse inhibition wasabout 46%, as compared to about 16% for the control group that receivedboth MK-801 and vehicle.

These results mean that this compound significantly ameliorates theschizophrenia-like MK-801-induced deficit in prepulse inhibition.

Elevated-Plus Maze

The goal of this experiment was to demonstrate the ability of the testcompounds to penetrate the CNS.

The experiment was conducted as follows.

Rats were tested in a standard elevated plus maze; testing occurred in adimly illuminated room using only two lights mounted over the maze.Animals were allowed to habituate to the room for at least one hourprior to treatment. One hour prior to testing, rats received a compoundof the present invention (0-30 mg/kg, P.O.). For testing, the rat wasplaced in the elevated plus maze for five minutes, alternating thestarting position between facing an open arm and facing a closed arm.The session was recorded and an observer blind to treatment recorded thenumber of explorations, entries and time spent in the open arm.Explorations were defined as the rat placing two feet into an open armwithout fully entering said arm. Entries were defined as the rat placingall four feet in an open arm. Time of entry in the open arm was recordedfrom the time the rat placed four feet in the open arm until two of therats′ feet entered the open square.

After administration of a compound of the present invention, the ratincreased the amount of time spend on the open arms of the maze,demonstrating a reduced anxiety and effective alleviation of symptomsassociated with schizophrenia. Specifically, as seen in FIG. 19, ratstreated with 10 or 30 mg/kg, PO (orally) of Pro-2023 spent around 27seconds on the open arms. This represents an increase of time spent onthe open arm of about 100%. As seen in FIG. 20, rats treated with 10mg/kg, PO (orally) of Pro-4047 spent around 22 seconds on the open armswhich is not a significant increase over control. This could be becausethe pharmacokinetics are such that the therapeutic window was missedwith the single time-point tested. In addition, this could be due to thedose-range tested, which may be sub-threshold doses or the sample sizeis too small to achieve statistical significance. As seen in FIG. 21,rats treated with 10 mg/kg, PO (orally) of Pro-4051 spent around 48seconds on the open arms or about a 150% increase over control. As seenin FIG. 22, rats treated with 10 mg/kg, PO (orally) of Pro-4051a spentaround 22 seconds on the open arms which is not a significant increaseover control. Thus, Pro-2023, and Pro-4051 demonstrate the ability toalleviate symptoms associated with schizophrenia in vivo.

Brain Levels of NAC and Glutathione Following Oral Administration

The goal of this experiment was to demonstrate the pharmacokineticproperties of test compounds in the brain of C57BL/6 mice.

The experiment was conducted as follows.

A compound of the invention was administered orally to C57BL/6 mice ateither 10 or 100 mg/kg. Brain samples were collected at 0.25, 0.50, 1, 2and 4 hours following the oral administration. Levels of NAC andglutathione were quantified in the brain samples using liquidchromatography-mass spectrometry (LC-MS/MS).

FIG. 23 depicts the levels of NAC found in the brain following oraladministration of Pro-2023. At 0.5 hours oral administration of 100mg/kg of Pro-2023 caused NAC to occur in the brain at about 2,000 pmol/gof brain tissue or about 1.25 times that of the vehicle (about 1,600pmol/g). Surprisingly, at 1 hour NAC remained in the brain at about1,900 pmol/g whereas the control fell back to about 1,400 pmol/g. Thisresult represents a 1.35 times difference between NAC and the control.

FIG. 24 depicts the levels of NAC found in the brain following oraladministration of Pro-2024. At 1.0 hours oral administration of 100mg/kg of Pro-2024 caused NAC to occur in the brain at about 1,800 pmol/gof brain tissue or about 1.29 times that of the vehicle (about 1,400pmol/g).

FIG. 25 depicts the levels of NAC found in the brain following oraladministration of Pro-4051. At 0.25 hours oral administration ofPro-4051 caused NAC to occur in the brain at about 4,500 pmol/g of braintissue or about 6 times that of the vehicle (about 750 pmol/g).Surprisingly, at the same time point, oral administration of Pro-4051caused NAC to occur in the brain about 3 times more than the oraladministration of NAC itself (about 1,500 pmol/g).

FIG. 26 depicts the levels of glutathione found in the brain followingoral administration of Pro-2023. NAC is known to elevate intracellularlevels of glutathione. At 4 hours oral administration of 100 mg/kg ofPro-2023 caused glutathione to occur in the brain at about 200 nmol/g ofbrain tissue or about 2 times that of the vehicle (about 95 nmol/g).

FIG. 27 depicts the levels of glutathione found in the brain followingoral administration of Pro-2024. At 4.0 hours oral administration of 10mg/kg of Pro-2024 caused glutathione to occur in the brain at about 190nmol/g of brain tissue or about at 2 times that of the vehicle (about 95nmol/g).

FIG. 28 depicts the levels of glutathione found in the brain followingoral administration of Pro-3010. At 4.0 hours oral administration of 100mg/kg of Pro-3010 caused glutathione to occur in the brain at about 195nmol/g of brain tissue or about at 2 times that of the vehicle (about 95nmol/g).

FIG. 29 depicts the levels of glutathione found in the brain followingoral administration of Pro-4051. At 4.0 hours oral administration of 100mg/kg of Pro-4051 caused glutathione to occur in the brain at about 1960nmol/g of brain tissue or about 1.05 times that of the vehicle (about1870 nmol/g).

These results demonstrate that oral administration of Pro-2023 orPro-4051 and possibly Pro-2024 are able to more effectively elevatelevels of NAC in the brain than the oral administration of NAC itselfand thus may be more effective than NAC for the treatment of CNSdiseases, such as schizophrenia, that currently respond to large dosesof NAC. In addition, these compounds, along with Pro-3010, havedemonstrated the ability to increase glutathione in the brain.

What is claimed is:
 1. A method of treating trichotillomania comprisingadministering a therapeutically effective amount of the compound offormula I

to a human in need thereof.
 2. The method of claim 1, wherein thecompound of formula I is administered orally.
 3. The method of claim 1,wherein the compound of formula I is administered rectally,parenterally, intracisternally, intravaginally, transdermally,transmucosally, sublingually, pulmonarily, intraperitoneally, topically,or bucally.
 4. The method of claim 1, wherein the compound of formula Iis administered in a solid form.
 5. The method of claim 1, wherein thecompound of formula I is administered in a capsule.
 6. The method ofclaim 1, wherein the compound of formula I is administered in a softgelatin capsule.
 7. The method of claim 1, wherein the compound offormula I is administered in a hard gelatin capsule.
 8. The method ofclaim 1, wherein the compound of formula I is administered as a tablet.9. The method of claim 1, wherein the compound of formula I isadministered in a solid form with an enteric coating.
 10. The method ofclaim 1, wherein the compound of formula I is administered in acomposition that releases the compound in a delayed manner.
 11. Themethod of claim 1, wherein the compound of formula I is administered ina composition that releases the compound in the intestinal tract. 12.The method of claim 1, wherein the compound of formula I is administeredin a liquid form.
 13. The method of claim 1, wherein from about 0.0001to about 2,000 mg/kg of the compound of formula I is administered to thesubject in one day.
 14. The method of claim 1, wherein from about 0.001to about 15 mg/kg of the compound of formula I is administered to thesubject in one day.
 15. The method of claim 1, wherein the compound offormula I is administered in a pharmaceutical composition containing apharmaceutically acceptable carrier.
 16. The method of claim 1, whereinthe therapeutically effective amount of the compound of formula I isadministered in multiple doses in a day.
 17. The method of claim 1,wherein the therapeutically effective amount of the compound of formulaI is administered in a single dose in a day.
 18. The method of claim 1,wherein the compound of formula I is the compound


19. A method of treating trichotillomania comprising administering atherapeutically effective amount of a salt or ester of the compound offormula I

to a human in need thereof.
 20. The method of claim 19, wherein a saltof the compound of formula I is administered.
 21. The method of claim20, wherein the salt is an organic amine.